Department of Environmental Medical Biology, Arthropods of Medical Importance Resource Bank, Institute of Tropical Medicine, Yonsei University College of Medicine, Seoul, Korea.
Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.
Microbiologyopen. 2021 Aug;10(4):e1220. doi: 10.1002/mbo3.1220.
High-throughput sequencing (HTS) of 16S rRNA gene amplicons provides compositional information regarding the microbial community, but not the absolute abundance of the bacteria. We aimed to develop a standardized method for quantifying the absolute abundance of bacteria in microbiome studies. To demonstrate the utility of our approach, we quantified the number of bacteria from the compositional data of the fecal and cecal microbiomes. The 16S rRNA gene of a hyperthermophile, Thermus aquaticus, was cloned into Pichia pastoris (yeast) genome, and an equivalent amount of the yeast was added to the stool and cecal samples of mice before DNA extraction. 16S rRNA gene library construction and HTS were performed after DNA extraction. The absolute abundances of bacteria were calculated using T. aquaticus reads. The average relative abundances of T. aquaticus in the five stool and five cecal samples were 0.95% and 0.33%, respectively, indicating that the number of bacteria in a cecum sample is 2.9 times higher than that in a stool sample. The method proposed for quantifying the absolute abundance of the bacterial population in this study is expected to overcome the limitation of showing only compositional data in most microbiome studies.
高通量测序（HTS）16S rRNA 基因扩增子可提供微生物群落的组成信息，但不能提供细菌的绝对丰度。我们旨在开发一种标准化方法来定量微生物组研究中细菌的绝对丰度。为了展示我们方法的实用性，我们从粪便和盲肠微生物组的组成数据中定量了细菌的数量。将嗜热菌 Thermus aquaticus 的 16S rRNA 基因克隆到毕赤酵母（酵母）基因组中，并在提取 DNA 之前将等量的酵母添加到小鼠的粪便和盲肠样本中。提取 DNA 后，进行 16S rRNA 基因文库构建和 HTS。使用 T. aquaticus 读数计算细菌的绝对丰度。在五个粪便和五个盲肠样本中，T. aquaticus 的平均相对丰度分别为 0.95%和 0.33%，这表明盲肠样本中的细菌数量是粪便样本的 2.9 倍。本研究中提出的定量细菌种群绝对丰度的方法有望克服大多数微生物组研究中仅显示组成数据的局限性。
